The ubiquitin conjugation system (UCS) plays a major role in the degradation of cellular proteins in eukaroytic cells and in some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of other important regulatory proteins that control gene transcription and cell cycle progression. The UCS is reported to degrade mitotic cyclic kinases, oncoproteins, tumor suppressors, viral proteins, transcriptional regulators, and receptors associated with signal transduction (Verma, R. et al. (1997) Science 278:455-460; Ciechanover, A. (1994) Cell 79:13-21).
There are several steps in the process of ubiquitin conjugation and protein degradation (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First ubiquitin (Ub), a small, heat stable protein is activated by a ubiquitin-activating enzyme (E1). This activation involves an ATP dependent binding of the C-terminus of Ub to the thiol group of an internal cysteine residue of E1. Then activated Ub is transferred to one of several Ub-conjugating enzymes (E2). Each E2 has a recognition subunit which allows it to interact with proteins carrying a particular degradation signal. E2 links the Ub molecule through its C-terminal glycine to an internal lysine of the target protein. It must be noted that different ubiquitin-dependent proteolytic pathways employ structurally similar, but distinct, E2s, and in some instances, accessory factors known as ubiquitin-ligases or E3s, are required to work in conjunction with E2s for recognition of certain substrates. More than one Ub molecule may be needed to ubiquinate a target protein which subsequently is recognized and degraded by a proteasome. After degradation, Ub is released and reutilized.
Prior to activation, Ub is usually expressed as a fusion protein composed of an N-terminal ubiquitin and a C-terminal extension protein (CEP) or as a polyubiquitin protein with Ub monomers attached head to tail. CEPs bear similarities to a variety of regulatory proteins in that most are highly basic, contain up to 30% lysine and arginine residues, and have nucleic acid-binding domains (Monia, B. P. et al. (1989) J. Biol. Chem. 264:4093-4103). The fusion protein is an important intermediate form which appears to allow co-regulation of the cell's translational and protein degradation activities and to localize inactive enzyme to specific cellular sites. Once delivered, C-termninal hydrolases cleave the fusion protein releasing Ub to carry out its work (Monia et al., supra).
The E2s are important for substrate specificity in different UCS pathways. All E2s have a conserved UBC domain of approximately 16 kD and at least 35% identity and contain a centrally located cysteine residue which is required for ubiquitin-enzyme thiolester formation (Jentsch, supra). A highly conserved proline-rich element is located N-terminal to the active cysteine residue. Structural variations beyond the conserved domain are used to classify the E2 enzymes. The E2s of class 1 (E2-1) consist almost exclusively of the conserved UBC domain and include yeast E2-1 and UBCs 4, 5, and 7. These E2s are thought to require E3 to carry out their activities (Jentsch, supra). UBC7 has been shown to recognize ubiquitin as a substrate and to form polyubiquitin chains in vitro (Van Nocker, S. et al. (1996) J. Biol. Chem. 271:12150-58). E2s of class II (E2-2) have various unrelated C-terminal extensions that contribute to substrate specificity and cellular localization. The yeast E2-2 enzymes, UBC2 and UBC3, have highly acidic C-terminal extensions that promote interactions with basic substrates such as histones. Yeast UBC6 has a hydrophobic signal-anchor sequence that localizes the protein to the endoplasmic reticulum.
Defects or alterations in the normal activity of the UCS are associated with a number of diseases and disorders. These include increased ubiquitin-dependent proteolysis as associated with cachexia (Llovera M. et al. (1995) Int. J. Cancer 61:138-141), degradation of the tumor-suppressor protein, p53 (Ciechanover, supra), and neurodegeneration such as observed in Alzheimer's disease (Gregori L. et al. (1994) Biochem. Biophys. Res. Commun. 203:1731-1738). Since ubiquitin conjugation is a rate-limiting step in antigen presentation, the ubiquitin degradation pathway may also have a critical role in the immune response (Grant E. P. et al. (1995) J. Immunol. 155:3750-3758).
The discovery of new ubiquitin-conjugating enzymes and the polynucleotides encoding them satisfies a need in the art by providing new compositions useful in the diagnosis, prevention and treatment of neoplastic, immune, developmental, and neuronal disorders.